BIOTECHNOLOGY 3 5 Genetic Modification and Biotechnology Human

BIOTECHNOLOGY 3. 5 Genetic Modification and Biotechnology

Human Genome Project A commitment by the scientific community to determine the location and structure of all genes in the human chromosomes Scientists sequence the genes of a section of a chromosome and pool the information together.

Having a map of the sequence of nucleotides of human DNA can lead to mapping of genes (listing and finding the locus of each human gene)

Human Genome Project: OUTCOME Improved understanding of genetic diseases Production of medicines (based on DNA sequences) to cure diseases Genetic screening (and preventative medicine) Focused research Provides more information about the evolutionary paths between species

Genetic Engineering/Modification The deliberate manipulation of genetic material It is possible to move genetic material from one species to another because the genetic code is universal ◦ All organisms use the same nitrogenous bases ◦ In all organisms, the same codon codes for the same amino acid (i. e. : in all species, AUG codes for the amino acid methionine)

Recombinant DNA Recombinant DNA: a fragments of DNA composed of sequences originating from at least 2 different sources Why? – ex: Agricultural Benefits: increase in crop yields; disease resistance; crop longevity

Genetic Engineering Hypothetically…. . If we could insert a gene into another organism’s genome (DNA), that organism would express that gene (make the protein the gene codes for) To do this, we would need “molecular scissors” to cut the gene sequence from our original source and “molecular glue” to insert the gene sequence into the host organism’s DNA

Restriction Endonucleases Also known as Restriction Enzymes are “molecular scissors” that can cleave (cut) double stranded DNA at a specific base-pair sequences. Bacterial enzymes Recognition Site: the specific sequence where the restriction enzyme makes its cut. ◦ Usually palindromic ◦ ~4 to 8 nucleotides long

STICKY ENDS

When a restriction enzyme cuts DNA at the recognition site to produces 2 fragments of DNA, the fragments can have either sticky ends or blunt ends. STICKY ENDS: both fragments of the newly cleaved DNA have DNA nucleotides lacking complimentary bases. BLUNT ENDS: The ends of the DNA fragments are fully paired.

Restriction enzymes that produce sticky ends are more useful because these DNA fragments can easily be joined to other DNA sticky ends fragments made by the same restriction enzyme. Can easily be used to create recombinant DNA

Technique for Gene Transfer Materials Required: ◦ DNA (with the gene you want to transfer) ◦ A vector (DNA used to carry the gene into the host cell) ◦ A host cell (which will express the gene – make the protein) ◦ Restriction enzymes ◦ DNA ligase

Technique for Gene Transfer to Bacteria 1. A bacterial plasmid (the vector) is obtained and the DNA with the desired gene are obtained. Bacteria carry their DNA in one large circular DNA strand However, they posses extra DNA in the form of plasmids – circular bits of genetic material carrying ~2 -30 genes

2. Both the plasmid and the DNA are exposed to the SAME the restriction enzyme. The restriction enzyme that is chosen must: ◦ Cut the plasmid only once. This mean the sequence of nucleotides that comprise the recognition site can only appear once in the entire plasmid ◦ NOT cut the plasmid at the origin of replication (this is where helicase attaches to the DNA) ◦ Cut the DNA with the desired gene twice. Once before the gene and once after. In most cases, the restriction enzyme used will produce sticky ends.

3. The gene that has been cut out is added to the plasmid with the help of DNA ligase Since the same restriction enzyme was used to cut both of them, they will have complimentary sticky ends. Following complimentary base pairing rules, the 2 fragments will bond together (with the help of DNA ligase) to create the recombinant plasmid / recombinant DNA

DNA Ligase is used to join the adjacent nucleotide cut sticky end fragments of DNA together T 4 DNA Ligase – is an enzyme from T 4 bacteriophage joins blunt ends together.

4. Now the recombinant plasmid can be inserted into the host cell (such as a harmless bacterial cell). The host cell will now be able to express the inserted gene (using their own cellular materials) Since the origin of replication on the plasmid is still intact, the recombinant plasmid will be replicated and will be passed on to the next generation.

Restriction sites The DNA that we want to insert the gene into The gene we want to introduce The gene is cut from its original DNA strand

Gene Transfer NOTE: It is actually easier to obtain messenger RNA transcripts of genes than the actual gene (from the DNA). Reverse transcriptase makes c. DNA (DNA copies of RNA molecules) and then that c. DNA can be inserted into a plasmid.

Animation http: //highered. mcgrawhill. com/sites/0072437316/student_view 0/c hapter 16/animations. html#

The real purpose of restriction enzymes Restriction enzymes act as an immune system in the bacterium. When a bacteriophage (a virus) tries to inject its DNA into the bacteria, restriction enzymes cut up the bacteriophage DNA into many fragments – thus, preventing it from doing any harm to the bacterium.

Methylases Restriction endonucleases must be able to distinguish between foreign DNA and their own DNA otherwise they would cut up their own DNA. METHYLASES are enzymes that modify a restriction site by adding a methyl group to and preventing the restriction endonuclease from cutting it. This prevents the cell from cutting its own DNA.

Real Life Application: INSULIN (p 121 -2) Insulin is a hormone made by the pancreas that regulates blood sugar levels by converting excess glucose into glycogen for energy storage. Diabetics do not make sufficient amounts of insulin. Diabetics may be required to take insulin injections to compensate.

Insulin continued Using restriction enzymes, the gene for synthesizing insulin is cleaved out of DNA and inserted into the DNA plasmid of a nonharmful bacteria. DNA ligase is added to the bacteria. The bacteria is also given the necessary amino acids.

Insulin Continued The recombinant DNA will express the insulin gene and make insulin (the DNA is transcribed into m. RNA, and then translated into the insulin protein) The insulin can be collected and administered to diabetics.

Did you know? ? ? How was diabetes treated before this technology? Diabetics were commonly given injections of animal insulin that is similar to human insulin ◦ Porcine insulin (from pigs) has only 1 amino acid differences ◦ Bovine insulin (from cattle) has 3 differences ◦ Shark insulin has 17 differences. Despite the differences, they all bind to the human insulin receptor and thus do the job

Did you know? ? ? Some diabetics are allergic to animal insulin so human insulin is preferable. In 1982, human insulin produced from genetically modified E. coli became commercially available. Since then methods using yeast cells and safflower plants have been developed.